Section

[Code of Federal Regulations]

[Title 40, Volume 31]

[Revised as of July 1, 2006]

From the U.S. Government Printing Office via GPO Access

[CITE: 40CFR799.9365]

[Page 372-381]

TITLE 40--PROTECTION OF ENVIRONMENT

CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)

PART 799_IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE

TESTING REQUIREMENTS--Table of Contents

Subpart H_Health Effects Test Guidelines

Sec. 799.9365 TSCA combined repeated dose toxicity study with the

reproduction/developmental toxicity screening test.

(a) Scope--(1) Applicability. This section is intended to meet

testing requirements of the Toxic Substances Control Act (TSCA) (15

U.S.C. 2601).

(2) Source. The source material used in developing this TSCA test

guideline is the Office of Prevention, Pesticides and Toxic Substances

(OPPTS) harmonized test guideline 870.3650 (July 2000, final

guidelines). This source is available at the address in paragraph (h) of

this section.

(b) Purpose. (1) This screening test provides limited information on

systemic toxicity, neurotoxicity, and/or immunotoxicity following

repeated exposure over a limited time period. In addition, it can be

used to provide initial information on possible effects on

[[Page 373]]

male and female reproductive performance such as gonadal function,

mating behavior, conception, development of the conceptus, and

parturition. It is not an alternative to, nor does it replace, the

existing test guidelines in Sec. Sec. 799.9370, 799.9380, 799.9620, and

799.9780 of this part.

(2) This test does not provide complete information on all aspects

of reproduction and development. In particular, it offers only limited

means of detecting postnatal manifestations of prenatal exposure, or

effects that may be induced during postnatal exposure. Due (amongst

other reasons) to the selectivity of the end points, and the short

duration of the study, this method will not provide evidence for

definite claims of no reproduction/developmental effects.

(3) This test can be used to provide initial information either at

an early stage of assessing the toxicological properties of chemicals,

or chemicals of high concern. It can also be used as part of a set of

initial screening tests for existing chemicals for which little or no

toxicological information is available or when otherwise considered

relevant. It also can serve as an alternative to conducting two separate

screening tests for repeated dose toxicity as described in Sec.

799.9305 of this part and reproductive/developmental toxicity as

described in Sec. 799.9355 of this part.

Part 792--Good Laboratory Practice Standards apply to this section. The

following definitions also apply to this section.

Dosage is a general term comprising dose, its frequency and the

duration of dosing.

Dose is the amount of test substance administered. Dose is expressed

as weight (g, gm) or as weight of test substance per unit weight of test

animal (e.g., mg/kg), or as constant dietary concentration (parts per

million (ppm)).

No-observed-effects level (NOEL) is the maximum dose used in a study

which produces no adverse effects. The NOEL is expressed in terms of the

weight of a test substance given daily per unit weight of test animal

(milligrams per kilogram per day).

(d) Principle of the test. (1) The test substance must be

administered in graduated doses to several groups of males and females.

Males should be dosed for a minimum of 4 weeks, up to and including the

day before scheduled sacrifice (this includes a minimum of 2 weeks prior

to mating, during the mating period and, approximately, 2 weeks post

mating). In view of the limited pre-mating dosing period in males,

fertility may not be a particularly sensitive indicator of testicular

toxicity. Therefore, a detailed histological examination of the testes

is essential. The combination of a pre-mating dosing period of 2 weeks

and subsequent mating/fertility observations with an overall dosing

period of at least 4 weeks, followed by detailed histopathology of the

male gonads, is considered sufficient to enable detection of the

majority of effects on male fertility and spermatogenesis.

(2) Females should be dosed throughout the study. This includes 2

weeks prior to mating (with the objective of covering at least two

complete oestrous cycles), the variable time to conception, the duration

of pregnancy and at least 4 days after delivery, up to and including the

day before scheduled sacrifice.

(3) Duration of study, following acclimatization, is dependent on

the female performance and is approximately 54 days, (at least 14 days

pre-mating, (up to) 14 days mating, 22 days gestation, 4 days

lactation).

(4) During the period of administration, the animals are observed

closely each day for signs of toxicity. Animals which die or are

sacrificed during the test are necropsied and, at the conclusion of the

test, surviving animals are sacrificed and necropsied.

(e) Description of the method--(1) Selection of animal species. This

test guideline is designed for use with the rat. If other species are

used, appropriate modifications will be necessary. Strains with low

fecundity or well-known high incidence of developmental defects should

not be used. Healthy virgin animals, not subjected to previous

experimental procedures, should be used. The test animals should be

characterised as to species, strain, sex,

[[Page 374]]

weight and/or age. At the commencement of the study the weight variation

of animals used should be minimal and not exceed 20% of the mean weight of each sex. Where the study is

conducted as a preliminary study to a long-term or a full-generation

study, preferably animals from the same strain and source should be used

in both studies.

(2) Housing and feeding conditions. (i) The temperature in the

experimental animal room should be 22 [deg]C (3[deg]). The relative humidity should be at least 30%

and preferably not exceed 70% other than during room cleaning. Lighting

should be artificial, the sequence being 12 hours light, 12 hours dark.

For feeding, conventional laboratory diets may be used with an unlimited

supply of drinking water. The choice of diet may be influenced by the

need to ensure a suitable admixture of a test substance when

administered by this method.

(ii) Animals may be housed individually or be caged in small groups

of the same sex; for group caging, no more than five animals should be

housed per cage. Mating procedures should be carried out in cages

suitable for the purpose. Pregnant females should be caged individually

and provided with nesting materials.

(3) Preparation of the animals. Healthy young adult animals must be

randomised and assigned to the treatment groups and cages. Cages should

be arranged in such a way that possible effects due to cage placements

are minimized. The animals must be uniquely identified and kept in their

cages for at least 5 days prior to the start of the study to allow for

acclimatisation to the laboratory conditions.

(4) Preparation of doses. (i) It is recommended that the test

substance be administered orally unless other routes of administration

are considered more appropriate. When the oral route is selected, the

test compound is usually administered by gavage; however, alternatively,

test compounds may also be administered via the diet or drinking water.

(ii) Where necessary, the test substance is dissolved or suspended

in a suitable vehicle. It is recommended that, wherever possible, the

use of an aqueous solution/suspension be considered first, followed by

consideration of a solution/emulsion in oil (e.g., corn oil) and then by

possible solution in other vehicles. For non-aqueous vehicles the toxic

characteristics of the vehicle must be known. The stability of the test

substance in the vehicle should be determined.

(f) Procedure--(1) Number and sex of animals. It is recommended that

each group be started with at least 10 animals of each sex. Except in

the case of marked toxic effects, it is expected that this will provide

at least eight pregnant females per group which normally is the minimum

acceptable number of pregnant females per group. The objective is to

produce enough pregnancies and offspring to assure a meaningful

evaluation of the potential of the substance to affect fertility,

pregnancy, maternal and suckling behaviour, and growth and development

of the F1 offspring from conception to day 4 post-partum. If

interim sacrifices are planned, the number should be increased by the

number of animals scheduled to be sacrificed before the completion of

the study. Consideration should be given to an additional satellite

group of five animals per sex in the control and the top dose group for

observation of reversibility, persistence or delayed occurrence of

systemic toxic effects, for at least 14 days post treatment. Animals of

the satellite groups must not be mated and, consequently, must not used

for the assessment of reproduction/developmental toxicity.

(2) Dosage. (i) Generally, at least three test groups and a control

group should be used. If there are no suitable general toxicity data

available, a range finding study may be performed to aid the

determination of the doses to be used. Except for treatment with the

test substance, animals in the control group should be handled in an

identical manner to the test group subjects. If a vehicle is used in

administering the test substance, the control group should receive the

vehicle in the highest volume used.

(ii) Dose levels should be selected taking into account any existing

toxicity and (toxico-) kinetic data available for the test compound or

[[Page 375]]

materials. It should also be taken into account that there may be

differences in sensitivity between pregnant and non-pregnant animals.

The highest dose level should be chosen with the aim of inducing toxic

effects but not death nor obvious suffering. Thereafter, a descending

sequence of dose levels should be selected with a view to demonstrating

any dosage related response and no adverse effects at the lowest dose

level. Two- to four-fold intervals are frequently optimum and addition

of a fourth test group is often preferable to using very large intervals

(e.g., more than a factor of 10) between dosages.

(3) Limit test. If an oral study at 1-dose level of at least 1000

mg/kg body weight/day or, for dietary administration, an equivalent

percentage in the diet, or drinking water (based upon body weight

determinations), using the procedures described for this study, produces

no observable toxic effects and if toxicity would not be expected based

upon data from structurally related compounds, then a full study using

several dose levels may not be considered necessary. The limit test

applies except when human exposure indicates the need for a higher dose

level to be used. For other types of administration, such as inhalation

or dermal application, the physical chemical properties of the test

substance often may dictate the maximum attainable exposure.

(4) Administration of doses. (i) The animals are dosed with the test

substance daily for 7 days a week. When the test substance is

administered by gavage, this should be done in a single dose to the

animals using a stomach tube or a suitable intubation cannula. The

maximum volume of liquid that can be administered at one time depends on

the size of the test animal. The volume should not exceed 1 ml/100 g

body weight, except in the case of aqueous solutions where 2 ml/100 g

body weight may be used. Except for irritating or corrosive substances

which will normally reveal exacerbated effects with higher

concentrations, variability in test volume should be minimized by

adjusting the concentration to ensure a constant volume at all dose

levels.

(ii) For substances administered via the diet or drinking water, it

is important to ensure that the quantities of the test substance

involved do not interfere with normal nutrition or water balance. When

the test substance is administered in the diet either a constant dietary

concentration (parts per million (ppm)) or a constant dose level in

terms of the animals' body weight may be used; the alternative used must

be specified. For a substance administered by gavage, the dose should be

given at similar times each day, and adjusted at least weekly to

maintain a constant dose level in terms of animal body weight.

(5) Experimental schedule. (i) Dosing of both sexes should begin 2

weeks prior to mating, after they have been acclimatized for at least 5

days. The study should be scheduled in such a way that mating begins

soon after the animals have attained full sexual maturity. This may vary

slightly for different strains of rats in different laboratories, e.g.,

Sprague Dawley rats 10 weeks of age, Wistar rats about 12 weeks of age.

Dams with offspring should be sacrificed on day 4 post-partum, or

shortly thereafter. In order to allow for overnight fasting of dams

prior to blood collection (if this option is preferred), dams and their

offspring need not necessarily be sacrificed on the same day. The day of

birth (viz. when parturition is complete) is defined as day 0 post-

partum. Females showing no-evidence of copulation are sacrificed 24-26

days after the last day of the mating period. Dosing is continued in

both sexes during the mating period. Males should further be dosed after

the mating period at least until the minimum total dosing period of 28

days has been completed. They are then sacrificed, or, alternatively,

are retained and continued to be dosed for the possible conduction of a

second mating if considered appropriate.

(ii) Daily dosing of the parental females should continue throughout

pregnancy and at least up to, and including, day 3 post-partum or the

day before sacrifice. For studies where the test substance is

administered by inhalation or by the dermal route, dosing should be

continued at least up to, and including, day 19 of gestation.

[[Page 376]]

(iii) Animals in a satellite group scheduled for follow-up

observations, if included, must not mated. They should be kept at least

for a further 14 days after the first scheduled sacrifice of dams,

without treatment to detect delayed occurrence, or persistence of, or

recovery from toxic effects.

(iv) The experimental schedule is given in the following figure 1.

[GRAPHIC] [TIFF OMITTED] TR15DE00.065

(6) Mating procedure. Normally, 1:1 (one male to one female) matings

should be used in this study. Exceptions can arise in the case of

occasional deaths of males. The female should be placed with the same

male until pregnancy occurs or 2 weeks have elapsed. Each morning the

females should be examined for the presence of sperm or a vaginal plug.

Day 0 of pregnancy is defined as the day a vaginal plug or sperm is

found. In case pairing was unsuccessful, re-mating of females with

proven males of the same group could be considered.

(7) Observations. (i) General clinical observations should be made

at least once a day, preferably at the same time(s) each day and

considering the peak period of anticipated effects after dosing. The

health condition of the animals should be recorded. At least twice daily

all animals must be observed for morbidity and mortality.

(ii) Once before the first exposure (to allow for within-subject

comparisons), and at least once a week thereafter, detailed clinical

observations should be made in all animals. These observations should be

made outside the home cage in a standard arena and preferably at the

same time, each day. They should be carefully recorded; preferably using

scoring systems, explicitly defined by the testing laboratory. Effort

should be made to ensure that variations in the test conditions are

minimal and that observations are preferably conducted by observers

unaware of the treatment. Signs noted should include, but not be limited

to, changes in skin, fur, eyes, mucous membranes, occurrence of

secretions and excretions and autonomic activity (e.g., lacrimation,

piloerection, pupil size, unusual respiratory pattern). Changes in gait,

posture and response to handling as well as the presence of clonic or

tonic movements, stereotypies (e.g., excessive grooming, repetitive

circling), difficult or prolonged parturition or bizarre behaviour

(e.g., self-mutilation, walking backwards) should also be recorded.

[[Page 377]]

(iii) At one time during the study, sensory reactivity to stimuli of

different modalities (e.g., auditory, visual and proprioceptive stimuli)

assessment of grip strength and motor activity assessment should be

conducted in five males and five females, randomly selected from each

group. Further details of the procedures that could be followed are

given in the respective references. However, alternative procedures than

those referenced could also be used. In males, these functional

observations should be made towards the end of their dosing period,

shortly before scheduled sacrifice but before blood sampling for

hematology or clinical chemistry. Females should be in a physiologically

similar state during these functional tests and should preferably be

tested during lactation, shortly before scheduled sacrifice. In order to

avoid hypothermia of pups, dams should be removed from the pups for not

more than 30 to 40 minutes. Examples of procedures for observation are

described in the references in paragraphs (h)(3), (h)(4), (h)(5),

(h)(6), and (h)(7) of this section.

(iv) Functional observations made once towards the end of the study

may be omitted when the study is conducted as a preliminary study to a

subsequent subchronic (90-day) or long-term study. In that case, the

functional observations should be included in this follow-up study. On

the other hand, the availability of data on functional observations from

this repeated dose study may enhance the ability to select dose levels

for a subsequent subchronic or long-term study.

(v) Functional observations may also be omitted for groups that

otherwise reveal signs of toxicity to an extent that would significantly

interfere with the functional test performance.

(vi) The duration of gestation should be recorded and is calculated

from day 0 of pregnancy. Each litter should be examined as soon as

possible after delivery to establish the number and sex of pups,

stillbirths, live births, runts (pups that are significantly smaller

than corresponding control pups), and the presence of gross

abnormalities.

(vii) Live pups should be counted and sexed and litters weighed

within 24 hours of parturition (day 0 or 1 post-partum) and on day 4

post-partum. In addition to the observations on parental animals,

described by paragraphs (f)(7)(ii) and (f)(7)(iii) of this section, any

abnormal behaviour of the offspring should be recorded.

(8) Body weight and food/water consumption. (i) Males and females

should be weighed on the first day of dosing, at least weekly

thereafter, and at termination. During pregnancy, females should be

weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day

0 or 1 post-partum), and day 4 post-partum. These observations should be

reported individually for each adult animal.

(ii) During pre-mating, pregnancy and lactation, food consumption

should be measured at least weekly. The measurement of food consumption

during mating is optional. Water consumption during these periods should

also be measured, when the test substance is administered by that

medium.

(9) Hematology. (i) Once during the study, the following

hematological examinations should be made in five males and five females

randomly selected from each group: hematocrit, hemoglobin concentration,

erythrocyte count, total and differential leucocyte count, platelet

count and a measure of blood clotting time/potential.

(ii) Blood samples should be taken from a named site. Females should

be in a physiologically similar state during sampling. In order to avoid

practical difficulties related to the variability in the onset of

gestation, blood collection in females may be done at the end of the

pre-mating period as an alternative to sampling just prior to, or as

part of, the procedure for sacrificing the animals. Blood samples of

males should preferably be taken just prior to, or as part of, the

procedure for sacrificing the animals. Alternatively, blood collection

in males may also be done at the end of the pre-mating period when this

time point was preferred for females.

(iii) Blood samples should be stored under appropriate conditions.

(10) Clinical biochemistry. (i) Clinical biochemistry determinations

to investigate major toxic effects in tissues and, specifically, effects

on kidney and liver, should be performed on blood

[[Page 378]]

samples obtained from the selected five males and five females of each

group. Overnight fasting of the animals prior to blood sampling is

recommended\1\. Investigations of plasma or serum must include sodium,

potassium, glucose, total cholesterol, urea, creatinine, total protein

and albumin, at least two enzymes indicative of hepatocellular effects

(such as alanine aminotransferase, aspartate aminotransferase and

sorbitol dehydrogenase) and bile acids. Measurements of additional

enzymes (of hepatic or other origin) may provide useful information

under certain circumstances.

---------------------------------------------------------------------------

\1\ For a number of measurements in serum and plasma, most notably

for glucose, overnight fasting would be preferable. The major reason for

this preference is that the increased variability which would inevitably

result from non-fasting, would tend to mask more subtle effects and make

interpretation difficult. On the other hand, however, overnight fasting

may interfere with the general metabolism of the (pregnant) animals,

disturbs lactation and nursing behaviour, and, particularly in feeding

studies, may disturb the daily exposure to the test substance. If

overnight fasting is adopted, clinical biochemical determinations should

be performed after the conduct of functional observations in week 4 of

the study.

---------------------------------------------------------------------------

(ii) Optionally, the following urinalysis determinations could be

performed in five randomly selected males of each group during the last

week of the study using timed urine volume collection; appearance,

volume, osmolality or specific gravity, pH, protein, glucose and blood

or blood cells.

(iii) In addition, studies to investigate serum markers of general

tissue damage should be considered. Other determinations that should be

carried out if the known properties of the test substance may, or are

suspected to, affect related metabolic profiles include calcium,

phosphate, fasting triglycerides and fasting glucose, specific hormones,

methemoglobin and cholinesterase. These need to be identified on a case-

by-case basis.

(iv) Overall, there is a need for a flexible approach, depending on

the observed and/or expected effect with a given compound.

(v) If historical baseline data are inadequate, consideration should

be given to determination of hematological and clinical biochemistry

variables before dosing commences.

(11) Pathology--(i) Gross necropsy. (A) All adult animals in the

study must be subjected to a full, detailed gross necropsy which

includes careful examination of the external surface of the body, all

orifices, and the cranial, thoracic and abdominal cavities and their

contents. Special attention should be paid to the organs of the

reproductive system. The number of implantation sites should be

recorded. Corpora lutea should be counted.

(B) The testes and epididymides of all adult males should be weighed

and the ovaries, testes, epididymides, accessory sex organs, and all

organs showing macroscopic lesions of all adult animals, should be

preserved.

from each group, the liver, kidneys, adrenals, thymus, spleen, brain and

heart should be trimmed of any adherent tissue, as appropriate and their

wet weight taken as soon as possible after dissection to avoid drying.

Of the selected males and females, the following tissues should also be

preserved in the most appropriate fixation medium for both the type of

tissue and the intended subsequent histopathological examination: all

gross lesions, brain (representative regions including cerebrum,

cerebellum and pons), spinal cord, stomach, small and large intestines

(including Peyer's patches), liver, kidneys, adrenals, spleen, heart,

thymus, thyroid, trachea and lungs (preserved by inflation with fixative

and then immersion), uterus, urinary bladder, lymph nodes (preferably 1

lymph node covering the route of administration and another one distant

from the route of administration to cover systemic effects), peripheral

nerve (sciatic or tibial) preferably in close proximity to the muscle,

and a section of bone marrow (or, alternatively, a fresh mounted marrow

aspirate).

(D) Formalin fixation is not recommended for routine examination of

testes and epididymides. An acceptable method is the use of Bouin's

fixative for these tissues. The clinical and other findings may suggest

the need to examine additional tissues. Also, any organs

[[Page 379]]

considered likely to be target organs based on the known properties of

the test substance should be preserved.

(E) Dead pups and pups sacrificed at day 4 post-partum, or shortly

thereafter, should, at least, be carefully examined externally for gross

abnormalities.

(ii) Histopathology. (A) Full histopathology should be conducted on

the preserved organs and tissues of the selected animals in the control

and high dose groups and all gross lesions. These examinations should be

extended to animals of other dosage groups if treatment-related changes

are observed in the high dose group.

(B) Detailed testicular histopathological examination (e.g., using

Bouin's fixative, paraffin embedding and transverse sections of 4-5

m thickness) should be conducted with special

emphasis on stages of spermatogenesis and histopathology interstitial

testicular cell structure. The evaluation should identify treatment-

related effects such as retained spermatids, missing germ cell layers or

types, multinucleated giant cells or sloughing of spermatogenic cells

into the lumen (the specifications for the evaluation are discussed in

paragraph (g)(2) of this section). Examination of the intact epididymis

should include the caput, corpus, and cauda, which can be accomplished

by evaluation of a longitudinal section. The epididymis should be

evaluated for leukocyte infiltration, change in prevalence of cell

types, aberrant cell types, and phagocytosis of sperm. Periodic acid-

Schiff (PAS) and hematoxylin staining may be used for examination of the

male reproductive organs. Histopathological examination of the ovary

should detect qualitative depletion of the primordial follicle

population.

performed on tissues and organs identified as showing effects in the

treated groups.

(g) Data and reporting--(1) Data. Individual animal data should be

provided. Additionally, all data should be summarised in tabular form,

showing for each test group the number of animals at the start of the

test, the number of animals found dead during the test or sacrificed for

humane reasons, the time of any death or humane sacrifice, the number of

fertile animals, the number of pregnant females, the number of animals

showing signs of toxicity, a description of the signs of toxicity

observed, including time of onset, duration, and severity of any toxic

effects, the types of histopathological changes, and all relevant litter

data.

(2) Evaluation of results. (i) The findings of this toxicity study

should be evaluated in terms of the observed effects, necropsy and

microscopic findings. The evaluation will include the relationship

between the dose of the test substance and the presence or absence,

incidence and severity of abnormalities, including gross lesions,

identified target organs, infertility, clinical abnormalities, affected

reproductive and litter performance, body weight changes, effects on

mortality and any other toxic effects.

(ii) Because of the short period of treatment of the male, the

histopathology of the testes and epididymides must be considered along

with the fertility data, when assessing male reproduction effects. The

use of historic control data on reproduction/development (e.g. for

litter size) where available may also be useful as an aid to the

interpretation of the study.

(iii) When possible, numerical results should be evaluated by an

appropriate and general acceptable statistical method. The statistical

methods should be selected during the design of the study. Due to the

limited dimensions of the study, statistical analysis in the form of

tests for ``significance'' are of limited value for many endpoints,

especially reproductive endpoints. Some of the most widely used methods,

especially parametric tests for measures of central tendency, are

inappropriate. If statistical analyses are used then the method chosen

should be appropriate for the distribution of the variable examined and

be selected prior to the start of the study.

(3) Test report. The test report must include the following

information:

(i) Test substance:

(A) Physical nature and, where relevant, physicochemical properties.

(B) Identification data.

[[Page 380]]

(ii) Vehicle (if appropriate): Justification for choice of vehicle,

if other than water.

(iii) Test animals:

(A) Species/strain used.

(B) Number, age and sex of animals.

(D) Individual weights of animals at the start of the test.

(iv) Test conditions:

(A) Rationale for dose level selection.

(B) Details of test substance formulation/diet preparation, achieved

concentration, stability and homogeneity of the preparation.

(D) Conversion from diet/drinking water test substance concentration

(parts per mission (ppm)) to the actual dose (mg/kg body weight/day), if

applicable.

(E) Details of food and water quality.

(v) Results (toxic response data by sex and dose):

(A) Time of death during the study or whether animals survived to

termination.

(B) Nature, severity and duration of clinical observations (whether

reversible or not).

(D) Food consumption and water consumption, if applicable.

(E) Sensory activity, grip strength and motor activity assessments.

(F) Hematological tests with relevant baseline values,

(G) Clinical biochemistry tests with relevant baseline values.

(H) Effects on reproduction, including information on mating/

precoital interval, fertility, fecundity and gestation duration.

(I) Effects on offspring, including number of pups born (live and

dead), sex ratio, postnatal growth (pup weights) and survival (litter

size), gross abnormalities and clinical observations during lactation.

(J) Body weight at termination and organ weight data for the

parental animals.

(K) Necropsy data, including number of implantations and number of

corpora lutea.

(L) Calculations of pre- and postimplantation loss.

(M) Detailed description of histopathological findings.

(N) Statistical treatment of results, where appropriate.

(vi) Discussion of results.

(vii) Conclusions.

(h) References. For additional background information on this test

guideline, the following references should be consulted. These

references are available for inspection at the TSCA Nonconfidential

Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M

St., NW., Washington, DC, 12 noon to 4 p.m., Monday through Friday,

except legal holidays.

(1) Mitsumori, K., Kodama, Y., Uchida, O., Takada, K., Saito, M.

Naito, K., Tanaka, S., Kurokawa, Y., Usami, M., Kawashima, K., Yasuhara,

K., Toyoda, K., Onodera, H., Furukawa, F., Takahashi, M. and Hayashi,

Y., (1994). Confirmation Study, Using Nitro-Benzene, of the Combined

Repeat Dose and Reproductive/ Developmental Toxicity Test Protocol

Proposed by the Organization for Economic Cooperation and Development

(OECD). Journal of Toxicology and Science, 19:141-149.

(2) Tanaka, S., Kawashima, K., Naito, K., Usami, M., Nakadate, M.,

Imaida, K., Takahashi, M., Hayashi, Y., Kurokawa, Y. and Tobe, M.

(1992). Combined Repeat Dose and Reproductive/Developmental Toxicity

Screening Test (OECD): Familiarization Using Cyclophosphamide.

Fundamental and Applied Toxicology, 18:89-95.

(3) Tupper D.E., Wallace R.B. (1980). Utility of the Neurologic

Examination in Rats. Acta Neurobiological Exposure, 40:999-1003.

(4) Gad S.C. (1982). A Neuromuscular Screen for Use in Industrial

Toxicology. Journal of Toxicology and Environmental Health, 9:691-704.

(5) Moser V.C., McDaniel K.M., Phillips P.M. (1991). Rat Strain and

Stock Comparisons Using a Functional Observational Battery: Baseline

Values and Effects of Amitraz. Toxicology and Applied Pharmacology,

108:267-283.

(6) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. (1979). A Method

for the Routine Assessment of Fore- and Hindlimb Grip Strength of Rats

and

[[Page 381]]

Mice. Neurobehavorial Toxicology, 1:233-236.

(7) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Reiter L.W.,

Tilson H.A., MacPhail R.C. (1991). Interlaboratory Comparison of Motor

Activity Experiments: Implication for Neurotoxicological Assessments.

Neurotoxicology and Teratology 13:599-609.

[65 FR 78793, Dec. 15, 2000]